Summary: The folding of proteins to their native structures is key to their function and malfunction in the cells of living organisms. Protein folding occurs on a high dimensionality and complex surface, whose features are often hidden in standard ensemble studies of protein folding. In this project, we will continue to improve and develop novel single molecule fluorescence methodologies to probe such complex features of protein folding. We will focus on understanding the folding properties of two amyloidogenic proteins, Sup 35 and -synuclein. The misfolding and aggregation of such amyloidogenic proteins are implicated in a host of diseases including Mad Cow and Parkinson's (-synuclein). Additionally, mounting evidence recently implicates the aggregation of these proteins in biologically constructive roles, such as acting as a protein-only genetic vehicle in yeast (Sup 35). Hence, a detailed understanding of the folding and dynamics of such proteins is very important from the point of view of human health. We will build on our strong foundation of single molecule investigations of biological folding to further develop and apply a suite of single molecule fluorescence methods, including single molecule FRET, polarization and correlation spectroscopy, in combination with novel and powerful microfluidic methods and protein engineering to gain insights into the folding of these proteins, both as monomeric species, as well as during the early stages of the aggregation process. Our studies will uncover whether these monomeric proteins (both understood to be intrinsically disordered) have elements of residual structure, how repeat sequences influence their folding and dynamics, and how other key cellular factors such as chaperones (for Sup 35) and binding to membranes (for -synuclein) influence the folding and structural dynamics of such proteins. Furthermore, by monitoring their folding properties during the early stages of aggregation, we aim to understand how these two processes are coupled within the context of this highly complex and heterogenous mixture of oligomeric species, insight that will be valuable in the understanding of the molecular and structural mechanisms of protein amyloidosis. Finally, these insights are anticipated to be extremely valuable in the design of therapeutic strategies to combat amyloid diseases.